Mingchun Liu

A DEMETER-like DNA demethylase governs tomato fruit ripening

Significance This work shows that active DNA demethylation governs ripening, an important plant developmental process. Our work defines a molecular mechanism, which has until now been missing, to explain the correlation between genomic DNA demethylation and fruit ripening. It demonstrates a direct cause-and-effect relationship between active DNA demethylation and induction of gene expression in fruits. The importance of these findings goes far beyond understanding the developmental biology of ripening and provides an innovative strategy for its fine control through fine modulation of epimarks in the promoters of ripening related genes. Our results have significant application for plant breeding especially in species with limited available genetic variation. In plants, genomic DNA methylation which contributes to development and stress responses can be actively removed by DEMETER-like DNA demethylases (DMLs). Indeed, in Arabidopsis DMLs are important for maternal imprinting and endosperm demethylation, but only a few studies demonstrate the developmental roles of active DNA demethylation conclusively in this plant. Here, we show a direct cause and effect relationship between active DNA demethylation mainly mediated by the tomato DML, SlDML2, and fruit ripening— an important developmental process unique to plants. RNAi SlDML2 knockdown results in ripening inhibition via hypermethylation and repression of the expression of genes encoding ripening transcription factors and rate-limiting enzymes of key biochemical processes such as carotenoid synthesis. Our data demonstrate that active DNA demethylation is central to the control of ripening in tomato.

Ethylene Control of Fruit Ripening: Revisiting the Complex Network of Transcriptional Regulation

The plant hormone ethylene controls fruit ripening through a complex network of transcriptional regulations and interplay between multiple signaling pathways. The plant hormone ethylene plays a key role in climacteric fruit ripening. Studies on components of ethylene signaling have revealed a linear transduction pathway leading to the activation of ethylene response factors. However, the means by which ethylene selects the ripening-related genes and interacts with other signaling pathways to regulate the ripening process are still to be elucidated. Using tomato (Solanum lycopersicum) as a reference species, the present review aims to revisit the mechanisms by which ethylene regulates fruit ripening by taking advantage of new tools available to perform in silico studies at the genome-wide scale, leading to a global view on the expression pattern of ethylene biosynthesis and response genes throughout ripening. Overall, it provides new insights on the transcriptional network by which this hormone coordinates the ripening process and emphasizes the interplay between ethylene and ripening-associated developmental factors and the link between epigenetic regulation and ethylene during fruit ripening.

Comprehensive Profiling of Ethylene Response Factor Expression Identifies Ripening-Associated ERF Genes and Their Link to Key Regulators of Fruit Ripening in Tomato

A small subset of ethylene response factor genes emerge as main actors in controlling fruit ripening via both ethylene-dependent and RIN/NOR-mediated mechanisms. Our knowledge of the factors mediating ethylene-dependent ripening of climacteric fruit remains limited. The transcription of ethylene-regulated genes is mediated by ethylene response factors (ERFs), but mutants providing information on the specific role of the ERFs in fruit ripening are still lacking, likely due to functional redundancy among this large multigene family of transcription factors. We present here a comprehensive expression profiling of tomato (Solanum lycopersicum) ERFs in wild-type and tomato ripening-impaired tomato mutants (Never-ripe [Nr], ripening-inhibitor [rin], and non-ripening [nor]), indicating that out of the 77 ERFs present in the tomato genome, 27 show enhanced expression at the onset of ripening while 28 display a ripening-associated decrease in expression, suggesting that different ERFs may have contrasting roles in fruit ripening. Among the 19 ERFs exhibiting the most consistent up-regulation during ripening, the expression of 11 ERFs is strongly down-regulated in rin, nor, and Nr tomato ripening mutants, while only three are consistently up-regulated. Members of subclass E, SlERF.E1, SlERF.E2, and SlERF.E4, show dramatic down-regulation in the ripening mutants, suggesting that their expression might be instrumental in fruit ripening. This study illustrates the high complexity of the regulatory network connecting RIN and ERFs and identifies subclass E members as the most active ERFs in ethylene- and RIN/NOR-dependent ripening.

The chimeric repressor version of an Ethylene Response Factor (ERF) family member, Sl-ERF.B3, shows contrasting effects on tomato fruit ripening.

Fruit ripening involves a complex interplay between ethylene and ripening-associated transcriptional regulators. Ethylene Response Factors (ERFs) are downstream components of ethylene signaling, known to regulate the expression of ethylene-responsive genes. Although fruit ripening is an ethylene-regulated process, the role of ERFs remains poorly understood. The role of Sl-ERF.B3 in tomato (Solanum lycopersicum) fruit maturation and ripening is addressed here using a chimeric dominant repressor version (ERF.B3-SRDX). Over-expression of ERF.B3-SRDX results in a dramatic delay of the onset of ripening, enhanced climacteric ethylene production and fruit softening, and reduced pigment accumulation. Consistently, genes involved in ethylene biosynthesis and in softening are up-regulated and those of carotenoid biosynthesis are down-regulated. Moreover, the expression of ripening regulators, such as RIN, NOR, CNR and HB-1, is stimulated in ERF.B3-SRDX dominant repressor fruits and the expression pattern of a number of ERFs is severely altered. The data suggest the existence of a complex network enabling interconnection between ERF genes which may account for the pleiotropic alterations in fruit maturation and ripening. Overall, the study sheds new light on the role of Sl-ERF.B3 in the transcriptional network controlling the ripening process and uncovers a means towards uncoupling some of the main ripening-associated processes.

A dominant repressor version of the tomatoSl-ERF.B3gene confers ethylene hypersensitivity via feedback regulation of ethylene signaling and response components

Ethylene Response Factors (ERFs) are downstream components of the ethylene signal transduction pathway, although their role in ethylene-dependent developmental processes remains poorly understood. As the ethylene-inducible tomato Sl-ERF.B3 has been shown previously to display a strong binding affinity to GCC-box-containing promoters, its physiological significance was addressed here by a reverse genetics approach. However, classical up- and down-regulation strategies failed to give clear clues to its roles in planta, probably due to functional redundancy among ERF family members. Expression of a dominant repressor ERF.B3-SRDX version of Sl-ERF.B3 in the tomato resulted in pleiotropic ethylene responses and vegetative and reproductive growth phenotypes. The dominant repressor etiolated seedlings displayed partial constitutive ethylene response in the absence of ethylene and adult plants exhibited typical ethylene-related alterations such as leaf epinasty, premature flower senescence and accelerated fruit abscission. The multiple symptoms related to enhanced ethylene sensitivity correlated with the altered expression of ethylene biosynthesis and signaling genes and suggested the involvement of Sl-ERF.B3 in a feedback mechanism that regulates components of ethylene production and response. Moreover, Sl-ERF.B3 was shown to modulate the transcription of a set of ERFs and revealed the existence of a complex network interconnecting different ERF genes. Overall, the study indicated that Sl-ERF.B3 had a critical role in the regulation of multiple genes and identified a number of ERFs among its primary targets, consistent with the pleiotropic phenotypes displayed by the dominant repression lines.

Auxin Response Factor SlARF2 Is an Essential Component of the Regulatory Mechanism Controlling Fruit Ripening in Tomato

Ethylene is the main regulator of climacteric fruit ripening, by contrast the putative role of other phytohormones in this process remains poorly understood. The present study brings auxin signaling components into the mechanism regulating tomato fruit ripening through the functional characterization of Auxin Response Factor2 (SlARF2) which encodes a downstream component of auxin signaling. Two paralogs, SlARF2A and SlARF2B, are found in the tomato genome, both displaying a marked ripening-associated expression but distinct responsiveness to ethylene and auxin. Down-regulation of either SlARF2A or SlARF2B resulted in ripening defects while simultaneous silencing of both genes led to severe ripening inhibition suggesting a functional redundancy among the two ARFs. Tomato fruits under-expressing SlARF2 produced less climacteric ethylene and exhibited a dramatic down-regulation of the key ripening regulators RIN, CNR, NOR and TAGL1. Ethylene treatment failed to reverse the non-ripening phenotype and the expression of ethylene signaling and biosynthesis genes was strongly altered in SlARF2 down-regulated fruits. Although both SlARF proteins are transcriptional repressors the data indicate they work as positive regulators of tomato fruit ripening. Altogether, the study defines SlARF2 as a new component of the regulatory network controlling the ripening process in tomato.

The tomato Ethylene Response Factor Sl‐ERF.B3 integrates ethylene and auxin signaling via direct regulation of Sl‐Aux/IAA27

Plant growth and development is coordinated by complex networks of interacting hormones, and cross-talk between ethylene and auxin signaling is essential for a wide range of plant developmental processes. Nevertheless, the molecular links underlying the interaction between the two hormones remain poorly understood. In order to decipher the cross-talk between the Ethylene Response Factor Sl-ERF.B3 and Sl-IAA27, mediating ethylene and auxin signaling, respectively, we combined reverse genetic approaches, physiological methods, transactivation experiments and electrophoretic mobility shift assays. Sl-ERF.B3 is responsive to both ethylene and auxin and ectopic expression of its dominant repressor version (ERF.B3-SRDX) results in impaired sensitivity to auxin with phenotypes recalling those previously reported for Sl-IAA27 downregulated tomato lines. The expression of Sl-IAA27 is dramatically reduced in the ERF.B3-SRDX lines and Sl-ERF.B3 is shown to regulate the expression of Sl-IAA27 via direct binding to its promoter. The data support a model in which the ethylene-responsive Sl-ERF.B3 integrates ethylene and auxin signaling via regulation of the expression of the auxin signaling component Sl-IAA27. The study uncovers a molecular mechanism that links ethylene and auxin signaling in tomato.

Overexpression of the class D MADS-box gene Sl-AGL11 impacts fleshy tissue differentiation and structure in tomato fruits

MADS-box transcription factors are key elements of the genetic networks controlling flower and fruit development. Among these, the class D clade gathers AGAMOUS-like genes which are involved in seed, ovule, and funiculus development. The tomato genome comprises two class D genes, Sl-AGL11 and Sl-MBP3, both displaying high expression levels in seeds and in central tissues of young fruits. The potential effects of Sl-AGL11 on fruit development were addressed through RNAi silencing and ectopic expression strategies. Sl-AGL11-down-regulated tomato lines failed to show obvious phenotypes except a slight reduction in seed size. In contrast, Sl-AGL11 overexpression triggered dramatic modifications of flower and fruit structure that include: the conversion of sepals into fleshy organs undergoing ethylene-dependent ripening, a placenta hypertrophy to the detriment of locular space, starch and sugar accumulation, and an extreme softening that occurs well before the onset of ripening. RNA-Seq transcriptomic profiling highlighted substantial metabolic reprogramming occurring in sepals and fruits, with major impacts on cell wall-related genes. While several Sl-AGL11-related phenotypes are reminiscent of class C MADS-box genes (TAG1 and TAGL1), the modifications observed on the placenta and cell wall and the Sl-AGL11 expression pattern suggest an action of this class D MADS-box factor on early fleshy fruit development.

Ethylene Response Factors (ERF) are differentially regulated by different abiotic stress types in tomato plants

Plants are sessile organisms, hence to face environmental constrains they developed strategies that rely on the activation of stress-response genes under the control of specific transcription factors. The plant hormone ethylene mediates physiological, developmental and stress responses through the activation of Ethylene Response Factors (ERFs) which belong to a large multigene family of transcription factors. While an increasing number of studies supports the involvement of ERFs in abiotic stress responses, so far the specific role of ERF family members in different abiotic stress conditions remains unexplored. The present work investigates the expression profile of a set of ERFs, representative of different ERF types, in tomato plants subjected to cold, heat, salt, drought and flooding conditions. The study revealed that a group of ERFs is preferentially associated with cold and heat stress responses while another set is expressed in response to salt, water and flooding stresses. Transactivation assays indicated that ERFs can regulate the expression of abiotic stress genes regardless of whether or not they harbor conserved GCC or DRE cis-elements in their promoter region. The outcome of the study provides clue on which ERFs should be targeted when aiming to improve adaptation to a particular stress type.

A novel tomato F-box protein, SlEBF3, is involved in tuning ethylene signaling during plant development and climacteric fruit ripening

Ethylene is instrumental to climacteric fruit ripening and EIN3 BINDING F-BOX (EBF) proteins have been assigned a central role in mediating ethylene responses by regulating EIN3/EIL degradation in Arabidopsis. However, the role and mode of action of tomato EBFs in ethylene-dependent processes like fruit ripening remains unclear. Two novel EBF genes, SlEBF3 and SlEBF4, were identified in the tomato genome, and SlEBF3 displayed a ripening-associated expression pattern suggesting its potential involvement in controlling ethylene response during fruit ripening. SlEBF3 downregulated tomato lines failed to show obvious ripening-related phenotypes likely due to functional redundancy among SlEBF family members. By contrast, SlEBF3 overexpression lines exhibited pleiotropic ethylene-related alterations, including inhibition of fruit ripening, attenuated triple-response and delayed petal abscission. Yeast-two-hybrid system and bimolecular fluorescence complementation approaches indicated that SlEBF3 interacts with all known tomato SlEIL proteins and, consistently, total SlEIL protein levels were decreased in SlEBF3 overexpression fruits, supporting the idea that the reduced ethylene sensitivity and defects in fruit ripening are due to the SlEBF3-mediated degradation of EIL proteins. Moreover, SlEBF3 expression is regulated by EIL1 via a feedback loop, which supposes its role in tuning ethylene signaling and responses. Overall, the study reveals the role of a novel EBF tomato gene in climacteric ripening, thus providing a new target for modulating fleshy fruit ripening.